Fibrinogen preparations enriched in fibrinogen with an extended alpha chain

ABSTRACT

The present invention relates to fibrinogen preparations enriched in α-extended fibrinogen. Compositions comprising such preparations show improved clotting properties compared to preparations based on HMW Fib which typically contain no or only low amounts of α-extended fibrinogen. In particular, clot formation time and the clot strength of a clot made by α-extended fibrinogen are improved. In addition, plasmin-mediated degradation of α-extended fibrinogen is reduced as compared to plasma derived fibrinogen.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/877,354, filed Oct. 7, 2015, which is a continuation of U.S.application Ser. No. 13/520,615, filed Sep. 13, 2012 (now abandoned),which is a U.S. National Phase Application of PCT InternationalApplication PCT/EP2011/050191, filed Jan. 7, 2011, which claims priorityto Netherlands (NL) Application No.: 10150391.0, filed Jan. 8, 2010.Each of the above-identified applications is incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to fibrinogen preparations and the use offibrinogen preparations in medical applications. In particular, itrelates to fibrinogen preparations which are enriched in fibrinogen withan extended alpha chain, to methods for producing it and to itsapplications.

BACKGROUND OF THE INVENTION

Fibrinogen is a soluble plasma glycoprotein which is synthesized in thehuman body primarily by liver parenchymal cells. It is a dimericmolecule, consisting of two pairs of three polypeptide chains designatedAα, Bβ and γ, which are connected by disulfide bridges. The threepolypeptide chains are encoded by three separate genes. The predominantform (HMW fib) harbors an Aα chain which is synthesized as a 625 aminoacid precursor and is present in fibrinogen found in blood plasma as a610 amino acids polypeptide chain, the Bβ chain contains 461 and the γchain 411 amino acids. The three polypeptides are synthesizedindividually from 3 mRNAs. Assembly of the three component chains (Aα,Bβ and γ) into its final form as a six-chain dimer (Aα, Bβ, γ)₂ occursin the lumen of the endoplasmic reticulum (ER).

Fibrinogen circulates in blood at high concentrations (1-2 g/L) anddemonstrates a high degree of heterogeneity. It is estimated that ineach individual about one million different fibrinogen moleculescirculate. Most of these variants, which account for just a smallportion of the total fibrinogen (in most cases not more than a fewpercents), differ in function and structure.

Proteolysis of the carboxy-terminal part of the Aα chain results inthree major circulating forms of fibrinogen having clearly differentmolecular weights. Fibrinogen is synthesized in the high-molecularweight form (HMW; molecular weight 340 kDa; the predominant form of Aαchains in the circulation contains 610 amino acids). The degradation ofone of the Aα chains gives rise to the LMW form (MW=305 kDa); the LMW′form (270 kDa) is the variant where both Aα chains are partiallydegraded at the carboxy-terminus. In blood of healthy individuals,50-70% of the fibrinogen is HMW, 20-50% is fibrinogen with one or twodegraded Aα chains (de Maat and Verschuur (2005) Curr. Opin. Hematol.12, 377). The HMW and LMW′ variants show distinct differences inclotting time and fibrin polymer structure (Hasegawa N, Sasaki S. (1990)Thromb. Res. 57, 183).

Well-known variants which are the result of alternative splicing are theso-called gamma prime (γ′) variant and the α-ext Fib or Fib420 variant.

The α-ext Fib or Fib420 variant, which has a molecular weight of 420kDa, accounts for 1-3% of the total circulating fibrinogen (de Maat andVerschuur (2005) Curr. Opin. Hematol. 12, 377). The extended α-ext Fibisoform is distinguished from the conventional α-chain of fibrinogen bythe presence of an additional 236 residue C-terminus globular domain dueto alternative splicing. Contradictary data on the function andcharacteristics of Fib420 is given in literature. Based on studies withplasma derived α-ext Fib, Applegate et al., Blood (2000) 95: 2297,concluded that the polymerization and cross-linking properties of α-extFib are not grossly different from plasma derived HMW Fib. They concludethat the additional C-domain has no effect on coagulation and theysuggest that the function of the domain may be to supportintegrin-mediated cell adhesion. In EP 1 495 051 it is suggested thatFib420 might be less sensitive to degradation and could have an effecton clot structure. However, no substantiation is given and there is nosuggestion as to whether the effect may be an enhancement ordeterioration in clot structure or strength. Mosesson et al. (Biophys.Chem. 112, 209: 2004) have studied the ultrastructure of clots that arebased on umbilical cord plasma-derived α-ext Fib. They reported that thefibers of α-ext Fib clots are thinner and more branched than those basedon HMW fibrinogen, but that they have the same periodicity thatcharacterizes all fibrin fibers. The authors suggest that the likelyfunction for the extended α-chain in α-ext Fib is to provide sites forinteraction with cellular integrins.

DETAILED DESCRIPTION

The present invention relates to a fibrinogen preparation which containsat least 95% (w/w) fibrinogen and wherein at least 10% of the fibrinogenis in the form of Fib420.

In the field, Fib₄₂₀ is also referred to as ‘α-ext Fib’, ‘fibrinogenwith an extended alpha chain’ or ‘fibrinogen₄₂₀’. In the presentcontext, these terms are used interchangeably. All these terms refer toa symmetrical molecule of the structure (Aα_(ext), Bβ, γ), wherein bothconventional fibrinogen α-chains, as found in HMW fib, have beenreplaced by extended chains.

In the present context, the term ‘fibrinogen preparation’ refers tofibrinogen in isolated form, for example to a plasma isolate or cellsupernatant isolate of fibrinogen. It may also refer to a syntheticpreparation of fibrinogen. A fibrinogen preparation according to theinvention preferably contains at least 65% w/w, at least 70% w/w, atleast 75% w/w, at least 80% w/w or at least 85% fibrinogen, based ontotal protein. More preferably, it is a pure preparation in whichsubstantially no contaminants, such as other proteins, are present andit contains at least 90% w/w, at least 95% w/w, at least 96% w/w, atleast 97% w/w or at least 98% fibrinogen, based on total protein. Mostpreferably, a fibrinogen preparation according to the inventioncomprises at least 99% w/w or at least 99.5% w/w fibrinogen, based ontotal protein. Such pure fibrinogen preparations are particularlysuitable to formulate compositions which are used in medicalapplications, such as compositions for wound therapy and surgicalrepair.

According to the invention, at least 10% w/w of the fibrinogen in thepreparation is in the form of Fib420. Preferably, at least 15% w/w, atleast 20% w/w, at least 25% w/w or at least 30% w/w of the fibrinogen isin the form of Fib420. More preferably, at least 40% w/w, at least 50%w/w, at least 60% w/w, at least 70% w/w, at least 80% w/w or at least90% w/w is in the form of Fib420. Most preferably, at least 95% w/w, atleast 99% w/w or all of the fibrinogen is in the form of Fib420.

One advantage of the fibrinogen preparation according to the inventionis that plasmin-mediated digestion of the fibrinogen preparation isslower than for plasma derived fibrinogen. Its enhanced resistance toplasmin digestion is likely to be beneficial for treatment of patientswho suffer from hyperfibrinolysis which is often seen in cases ofacquired coagulopathy.

Using a fibrinogen preparation according to the invention, clots areformed faster and the clots which are formed have a higher clot firmnessthan clots formed with plasma derived fibrinogen or HMW fibrinogen. Thismeans that fibrin clot stability is enhanced when using the fibrinogenpreparation according to the invention and that the fibrinogenpreparation according to the invention is more potent than state of theart preparations. A more potent fibrinogen preparation allows for thereduction of both the amount of fluid and the amount of therapeuticalprotein to be administered. This is an advantage for intravenous use totreat dilutional coagulopathy. Currently, for intravenous (i.v)injection of fibrinogen to compensate for low blood clotting activity,high dosages of fibrinogen are required (˜5 gram of fibrinogen pertreatment). This is administered by direct injection at a dose of 70mg/kg. As fibrinogen in general can be dissolved at a maximumconcentration of 20 mg/ml, this means that about 250 ml of fluid has tobe administered intravenously to in an adult. Lowering this dose byproviding a more potent fibrinogen would be beneficial.

Yet another advantage is that clot formation using the fibrinogenpreparation of the invention is less Factor XIII dependent thanpreparations of plasma derived fibrinogen or HMW fibrinogen. Thestrength of a clot made in buffer by alpha-extended Fib (so in theabsence of Factor XIII) is higher than for plasma-derived fibrinogenwhich will contain some Factor XIII No Factor XIII is required to formfirm clots. Therefore, in one embodiment, the fibrinogen preparationaccording to the invention is free of Factor XIII.

Clotting time (CT), clot formation time (CFT) and clot firmness of α-extrhFib and plasma derived fibrinogen can be measured using ROTEManalysis. ROTEM® (Pentapharm GmbH, Munich, Germany) stands for ROtationThromboElastoMetry. The technique utilizes a rotating axis submerged ina (blood) sample in a disposable cuvette. Changes in elasticity underdifferent clotting conditions result in a change in the rotation of theaxis, which is visualized in a thromboelastogram, reflecting mechanicalclot parameters (see e.g. Luddington R. J. (2005) Clin Lab Haematol.2005 27(2):81).

In a ROTEM® system test under similar conditions, a fibrinogenpreparation according to the invention typically performs at least asgood or better than a fibrinogen preparation or composition in which thefibrinogen variant distribution resembles the variant distribution inhuman plasma, viz. less than 5% w/w of the fibrinogen is of the Fib420type. This is for example the case for fibrinogen concentrates which arebased on plasma-derived fibrinogen. Such concentrates are commerciallyavailable. In particular, its clot formation time will be less than theclot formation time of a fibrinogen preparation in which less than 5%w/w of the fibrinogen is of the Fib420 type. Preferably, the clotformation time of a fibrinogen preparation according to the invention ismaximally 80%, maximally 60%, maximally 50%, maximally 40%, maximally30%, maximally 20%, maximally 10% of the clot formation time of afibrinogen preparation in which less than 5% w/w of the fibrinogen is ofthe Fib420 type, such as plasma-derived fibrinogen preparations. TheROTEM experiments with α-ext rhFib presented in the Examples indicatethat a fibrinogen preparation according to the invention has a clotformation time (CFT) which is less than the CFT of purified plasmaderived fibrinogen produced for intravenous applications.

In another aspect, the invention relates to a composition comprising afibrinogen preparation according to the invention. In addition to thefibrinogen, the composition may comprise an activator, such as thrombinor a thrombin-like protein, such as reptilase. It may also compriseexcipients which are suitable for use in an injectable preparation. Thepreparation may be in dry form and subsequently reconstituted, with e.g.buffered saline, and the like, or it may be in liquid form, either as asuspension or solution. Suitable excipient materials may includesolvents and co-solvents, such as ethanol, glycerin, PEGs, oils, and thelike; solubilizing, wetting, suspending, emulsifying or thickeningagents, such as carboxymethylcellulose, hydrolyzed gelatin, pluronics,polysorbates, and the like; chelating agents, such as calcium EDTA,DTPA, and the like; antioxidants and reducing agents, such as BHT,ascorbic acid, sodium metabisulphite, and the like; antimicrobialpreservatives, such as benzyl alcohol, phenol, parabens, and the like;buffers and pH adjusting agents, such as tromethamine, sodium phosphate,sodium acetate, sodium hydroxide, and the like; bulking agents,protectants, and tonicity adjustors, such as alanine, albumin, dextran,lactose, sorbitol, sodium chloride, histidine, and the like; specialadditives, such as simethicone as anti-foaming agent, trehalose forreduction of protein aggregation.

The composition may be used in any application in which plasma-derivedor recombinant fibrinogen is used. The main applications are hemostasisand to seal tissue. In one embodiment of the invention, the compositionis a pharmaceutical composition. The pharmaceutical compositioncomprises a fibrinogen preparation according to the invention and apharmaceutically acceptable carrier. “Pharmaceutically acceptablecarrier” refers to a vehicle, auxiliary agent, adjuvant, diluent,excipient or carrier with which the fibrinogen preparation of theinvention is administered. Examples of pharmaceutically acceptablecarriers include, without limitation, water, buffered saline, ethanol,polyol (for example, glycerol, propylene glycol, liquid polyethyleneglycol and the like), dimethyl sulfoxide (DMSO), oils, detergents,suspending agents or suitable mixtures thereof. Suitablepharmaceutically acceptable carriers and formulations are described inRemington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co.,Easton, 1995) and “Remington: The Science And Practice Of Pharmacy” byAlfonso R. Gennaro (Lippincott Williams & Wilkins, 2005).

The pharmaceutical composition may be applied topically and may includecarriers which are water-soluble, water-absorbent, water-insoluble orwater-swellable. Suitable materials include saccharides such as mono-anddi-saccharides, including lactose, mannitol and trehalose, or dextranand dextran polymers, like e.g. Sephadex, which are available indifferent particle sizes, starches, pullulan derivatives, hyaluronicacid esters, and the like. Cellulose products such as microcrystallinecellulose (Avicel range), methylcellulose, carboxymethyl cellulose,microtine cellulose or hydroxy propyl cellulose, and other materialssuch as cross-linked polyvinyl pyrrolidone (PVP), may be used singly orin admixture. Also, suitable carriers include polyethylene glycol (PEG),preferably having a molecular weight of about 1000; polyvinylpyrrolidone(PVP), preferably having an average molecular weight of about 50,000;poly(acrylic acid), PVA, poly(methylvinylether co-maleic anhydride),poly(ethyleneoxide), and dextran, typically having an average molecularweight of about 40,000.

Tablet disintegrants may be included. These materials will absorbmoisture from the wound, expand rapidly and thereby enhance thewettability of the hemostatic components of the powder blend. Suitableexamples include sodium starch glycolate (Explotab® or Primojel®) whichhas an average particle size in the range of 35-55 μm. About 25% of theglucose units are carboxymethylated; cross-linked polyvinyl pyrrolidone(Polyplasdone®); alginates and alginic acid; cross-linked sodiumcarboxymethylcellulose (Ac-Di-Sol). Gums and gelling agents that can beused include, for example, tragacanth, karaya gum, soluble starch,gelatin, pectin, guar gum and gellan gum.

A particularly preferred additive is Emdex®, i.e. a hydrated form ofdextrates (spray crystallized dextrose containing small amounts ofstarch oligosaccharides). It is a highly refined product composed ofwhite, free-flowing, spray-crystallized macroporous spheres with amedian particle size of 190-220 μm.

A most preferred additive is NON-PAREIL SEEDS®: (Sugar Spheres). Theseare used in multiple drug units for improved content uniformity,consistent and controlled drug release and high drug stability, sizeranges from 200 to 2000 mm.

The pharmaceutically acceptable carrier may comprise an effervescentcouple. The gas produced following an effervescent reaction can expandthe fibrin sealant into a ‘foam’ and/or increase wettability of thepowders comprising the fibrin sealant. As the powders are applied to awound, the effervescent components dissolve, react and liberate, say,carbon dioxide, thereby increasing the wettability of the hemostaticcomponents and thus enhancing time to clot formation. The fibrin sealantwill appear as a stable foam once fully reacted and the clot has formed.

The effervescent couple typically comprises citric acid or sodiumhydrogen citrate and sodium bicarbonate, but other physiologicallyacceptable acid/alkaline or alkaline earth metal carbonate mixtures maybe used, for example tartaric, adipic, fumaric or malic acids, andsodium, potassium or calcium (bi)carbonates or sodium glycine carbonate.

In general it has been found that preferred taste characteristics areexhibited when the relative proportions of the components of theeffervescent couple on a chemical molecular equivalent basis are in therange of 4:3 to 1:3, more preferably about 2:3, expressed as the ratioof molecular equivalent of the acidic component to the basic component.In terms of a preferred combination of citric acid and sodiumbicarbonate these values represent on a weight basis, a range from 1:1to 0.3:1, preferably 0.5:1 expressed as the ratio of acidic to basiccomponent.

The pharmaceutical composition according to the invention is suitablefor facilitating tissue adherence, improving wound healing or forintravenous administration.

In one embodiment, the pharmaceutical composition according to theinvention is a fibrin sealant. The fibrin sealant according to theinvention typically comprises thrombin. The sealant may be in anyconvenient form, be it dry or liquid. A suitable example of a drysealant in which the fibrinogen preparation according to the inventionmay be used is Fibrocaps® powder sealant, which is described inWO97/44015 and which is based on micro-particles of fibrinogen andthrombin. The separate components are prepared by spray-drying,fibrinogen with trehalose and thrombin with trehalose. Each powdercomponent has a predominant particle size of approximately up to 50 μmin diameter. The Fibrocaps® fibrin sealant, which is a blend of thesecomponents, has been demonstrated to be an easy-to-use, stable andefficacious topical haemostat. The product can be used immediately,without reconstitution and is useful in wound therapy, in surgicalrepair and as an extravascular stent. On contact with aqueous fluid suchas blood, the exposed active thrombin converts the exposed fibrinogeninto insoluble fibrin polymers. The fibrinogen preparation according tothe invention would give further improved blood clotting properties.

The composition of the invention may be applied to wounds, sutures,incisions and other openings where bleeding may occur. A wound includesdamage to any tissue in a living organism. The tissue may be an internal(e.g. organ) or external tissue (e.g. eye or skin), and may be a hardtissue (e.g. bone) or a soft tissue (e.g, liver or spleen). The woundmay have been caused by any agent, including infection, surgicalintervention, burn or trauma. The composition of the invention may beused for surgical interventions such as in the gastrointestinal system,e.g. the oesophagus, stomach, small intestine, large intestine, rectum,on parenchymal organs such as the liver, pancreas, spleen, lungs,kidney, adrenal glands, lymph and thyroid glands; surgical interventionsin the ear, nose and throat area (ENT) including dental surgery,cardiovascular surgery, aesthetic surgery, neurological surgery,lymphatic, biliary, and cerebrospinal (C SF) fistulae, air leakagesduring thoracic and pulmonary surgery, thoracic surgery includingsurgery of the trachea, bronchi and lungs, gynaecological, vascular,urological, bone (e.g. spongiosa resection), and emergency surgery.

As an extravascular stent or support, the composition of the inventionmay be applied to the outside of a segment or the whole of a vein graft.A suitable extravascular stent composition is a composition of afibrinogen preparation according to the invention and thrombin. Thecomposition may be in any suitable form, be it liquid or dry. In oneembodiment, a dry powder composition, for instance one as describedabove, is used as an extravascular stent. The dry powder compositionpolymerizes in the limited amounts of bodily fluids which are naturallypresent at the outside of the vessel wall, thus forming an extravascularstent.

A vein coated with an extravascular stent composition according to thepresent invention is also part of the invention. The vein may be anykind of vein which needs to be protected from overextension or whichneeds support, for instance a varicose vein. In a preferred embodiment,the vein is a venous graft. In one embodiment, the composition isapplied before the venous graft is introduced in the human or animalbody. In another embodiment, the composition is applied after the venousgraft has been introduced in the human or animal body.

In yet another aspect, the present invention relates to the use of afibrinogen preparation according to the invention as a medicament. Itcan be used for the preparation of a medicament for the treatment ofacute bleeding episodes, hyperfibrinolysis, fibrinogen deficiency, be itacquired or congenital, or other bleeding disorders.

In one embodiment, a composition according to the invention is used in amethod for reducing bleeding at a hemorrhaging site. Preferably, ahemostatically effective amount of the composition according to theinvention is used. When used as a topical haemostat, a time tohemostasis (TTH) of about 10 minutes or less, about 5 minutes or less,or about 3 minutes or less may be achieved. In the present context, TTHis the time it takes to stop a bleeding. If a pressure sheet is used,measurement of TTH typically starts when the pressure sheet is appliedto the bleeding site and runs until bleeding stops by visualization ofthe dressing and/or no bleeding through or around the dressing isobserved.

In yet another aspect, the present invention relates to a method forpreparing a fibrinogen preparation according to the invention. Suchpreparation may be prepared in any suitable way, using techniquesavailable in the art. In order to produce α-ext Fib in an economicallyfeasible way, high expression levels of intact, functional fibrinogenare required and therefore recombinant production is preferred. In thecontext of the present invention, fibrinogen or a fibrinogen chain is‘in intact form’ when the amino acid sequence contains all the aminoacids which were encoded for by the nucleotide sequence, optionallywithout the amino acids which are removed during normal cell (secretion)processing. Therefore, alpha-ext chains having 866 or 847 amino acidsare examples of an alpha chain in intact form.

Recombinant production of fibrinogen has many advantages over the use ofplasma derived materials. These include its preferred safety profile,the possibility to make pure homogeneous preparations of variants freeof any other blood born contaminants and the unlimited supply. Inaddition, for specific applications (e.g. use of fibrinogen as anintravenous hemostat) proper post-translational modifications (e.g.glycosylation) are required. Therefore, expression in eukaryotic, inparticular mammalian systems, more particular in human systems, ispreferred.

In a preferred embodiment, a fibrinogen preparation according to theinvention is prepared by a method which comprises the steps of:

providing an expression vector comprising a nucleic acid sequence, whichnucleic acid sequence encodes an alpha extended polypeptide chain offibrinogen;

transforming a eukaryotic cell with the expression vector;

maintaining the transformed eukaryotic cell under such conditions whichallow for the expression of the nucleic acid sequence encoding the alphaextended polypeptide chain of fibrinogen.

Expression vectors for eukaryotic hosts are known in the art and any ofthe vectors conventionally used for expression in eukaryotic cells maybe used. An expression vector typically contains a promoter operablylinked to the nucleic acid sequence to be expressed and ribosome bindingsites, polyadenylation signals, transcription termination sequences,upstream regulatory domains and enhancers. In one embodiment, anexpression vector for expression in mammalian cells, such as for CHO orPER.C6® cells is used. Such vectors are known in the art and suitableexamples include pcDNA3.1 plasmids, GATEWAY (Invitrogen), pCMV/Bsd(Invitrogen), pFN vectors (Promega) and numerous other vector systems.The PER.C6® cells are as deposited under European Collection of AnimalCell Cultures (ECACC) No. 96022940.

In the present context, ‘an alpha extended polypeptide chain offibrinogen’ may refer to a fibrinogen alpha chain of 866 amino acidswith a signal sequence or to one without a signal sequence and to anyvariants thereof which have arisen through genetic polymorphisms ordifferences in glycosylation and phosphorylation. A suitable example ofan alpha extended chain amino acid sequence is given in SEQ ID No. 1.The terms ‘alpha chain’ and ‘Aα chain’ are used interchangeably in thecontext of the present invention.

The skilled person will understand that the eukaryotic cell must alsocontain nucleic acid sequences which encode the beta and gamma chain offibrinogen to be able to produce a fibrinogen molecule. Recombinantproduction of fibrinogen from alpha, beta and gamma chains have beendescribed before, see for instance PCT/EP2009/058754, U.S. Pat. No.6,037,457 or WO 95/023868.

In the context of the present invention, the terms ‘beta chain’ and ‘Bβchain’ are used interchangeably. They may refer to both wild type andvariants of the beta chain, with or without signal sequence. A suitableexample of a fibrinogen beta chain amino acid sequence is given in SEQID No. 2.

In the context of the present invention, the term ‘gamma chain’ and ‘γchain’ are used interchangeably. They may refer to both wild type andvariants of the gamma chain, with or without signal sequence. Suitableexamples of a fibrinogen gamma chain amino acid sequence are given inSEQ ID No. 3 and 4.

Preferably, the nucleic acid sequences encoding a fibrinogen chain areoptimized. An optimized nucleic acid sequence allows for the efficientexpression of recombinant fibrinogen in intact form. Preferably, theyare optimized for expression in a eukaryotic cell culture system, suchas for expression in a COS cell, BHK cell, NS0 cell, Sp2/0 cell, CHOcell, a PER.C6 cell, a HEK293 cell or insect cell culture system. Morepreferably, they are optimized for expression in a mammalian cellculture system. Most preferably, the nucleic acid sequences areoptimized for expression in a human cell culture system, such as for aPER.C6 cell or a HEK293 cell culture system. The nucleotide sequencewhich is optimized may be DNA or RNA. Preferably, it is cDNA.

An optimized nucleotide sequence encoding a fibrinogen alpha extended,beta or gamma chain shows at least 70% identity to its respectivenon-optimized counterpart. In one embodiment, an optimized nucleotidesequence encoding a fibrinogen α-ext Fib, Bβ and γ chain shows 70-80%identity to its respective non-optimized sequences. Preferably, theoptimized nucleotide sequences encoding a fibrinogen alpha extended,beta or gamma chain contain no cis-acting sites, such as splice sitesand poly(A) signals.

An optimized nucleotide sequence which is used in the method accordingto the invention and which encodes a fibrinogen alpha extended chaincontains no 39 basepair direct repeat sequences which are normallypresent in the gene encoding the alpha chain of human fibrinogen. Therepeating sequence must be changed without changing the encoded proteinsequence.

In a preferred embodiment, an optimised nucleotide sequence according toSEQ ID NO. 5 or the part of this sequence without the signal(nucleotides 60-2598) is used for expressing the alpha extended chain.

In another preferred embodiment, an optimised nucleotide sequenceaccording to SEQ ID NO. 6 or the part of this sequence without thesignal sequence (nucleotides 93-1473) is used for expressing the betachain.

In another preferred embodiment, an optimised nucleotide sequenceaccording to SEQ ID NO. 7 or 8 or the part of these sequences without asignal sequence (nucleotides 51-1311 of SEQ ID NO. 7 and nucleotides51-1359 of SEQ ID NO. 8) is used for expressing the gamma chain.

Nucleic acid sequences according to the invention may be encoding anytype of fibrinogen chains. Preferably they are encoding mammalianfibrinogen chains, more preferably they are encoding primate fibrinogenchains, most preferably they are encoding human fibrinogen chains. Alsocombinations are possible, such as for example one or two mammalianfibrinogen chains combined with two or one rodent fibrinogen chains.Recombinant expression according to the method of the present inventionallows for expression levels of Fib420 which are similar to those ofrecombinant HMW Fib.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Western blot analysis. Lane 1 is a control containing plasmaderived wild-type fibrinogen (FIB3, Enzyme Research Laboratories). Lane2 contains culture supernatant of clone W115, a PER.C6 clones expressingα-ext rhFib. The molecular weight marker (MW) is indicated at the left.

FIG. 2 ROTEM analysis. Clotting time, clot formation time and clotfirmness were determined by ROTEM analysis. The left panels display thefibrinogen preparations in buffer, the right panels display plasma mixed1:1 with the fibrinogen preparations. FIG. 2 includes Panels A throughF, as follows:

Panel A: Plasma derived fibrinogen (Haemocomplettan, CSL Behring,Marburg, Germany) in buffer;

Panel B: PER.C6 derived recombinant HMW fibrinogen (α-625) in buffer;

Panel C: PER.C6 derived rhFib-ext fibrinogen (a-847) in buffer;

Panel D: Plasma derived fibrinogen (Haemocomplettan, CSL Behring,Marburg, Germany) mixed with plasma;

Panel E: PER.C6 derived recombinant HMW fibrinogen (α-625) mixed withplasma;

Panel F: PER.C6 derived rhFib-ext fibrinogen (α-847) mixed with plasma.

FIG. 3 The picture shows a Coomassie stained protein gel loaded withmaterial from plasmin degraded plasma derived material (ERL FIB3; upperpanel, and α-ext Fib (lower panel). The conditions are indicated on topof the gel; the time of incubation (0, 1, 5, 30, 60 and 120 minutes ando/n (overnight)) is shown as well. At the right of the gel the MW markeris displayed.

EXAMPLES Example 1 Preparation of Optimized cDNA Constructs

cDNAs coding for human fibrinogen polypeptide chains of α-ext Fib(Fib420), Bβ and γ, were synthesized in codon optimized format byGeneArt (Regensburg, Germany): (i) cis-acting sites (splice sites,poly(A) signals) were removed; (ii) repeat sequence of Aα chain wasmodified; (iii) GC content was increased for prolonged mRNA half life;(iv) Codon usage was adapted to CHO (codon adaption index—CAI->0.95).

The codon optimized cDNAs for α-ext Fib (SEQ ID NO.5), Bβ (SEQ ID No.6), and γ (SEQ ID NO. 6) chain were subcloned in pcDNA3.1 deriviates.Aα-extended (Fib420) in pcDNA3.1(+) neo, Bβ chains in pcDNA3.1(+)hygroand γ chain in pcDNA3.1(−)hygro (Invitrogen, Carlsbad, USA).

Example 2 PER.C6 Cell Lines Expressing Recombinant Human α-ext Fib

The generation of PER.C6 cell lines producing recombinant humanfibrinogen is similar as described before in PCT/EP2009/058754. Insummary, cells were cultured in suspension in MAb medium and transfectedusing the AMAXA nucleofection device (program A-27) and usingNucleofector kit T with three vectors encoding the three differentchains of the human fibrinogen protein (Aα-ext, Bβ and γ chain) andcontaining the optimized cDNA chains (SEQ ID no.5, SEQ ID no. 6, and SEQID no.7, respectively).

After transfection and plating in 96-well plates, 325 clones weretransferred and screened in 48-well plates. At the end of the expansionpath, 24 clones were transferred to shaker flasks, of which 8 wereselected for stability and expression analysis in continued batchculture testing.

Yields in batch culture were similar to yields obtained with cell linesthat express the Aα-chain in 610 or 625 amino acid format, indicatingthat the extension of the Aα-chain does not impair expression levels.This was not expected on forehand, as plasma derived fibrinogen onlycontains 1-3% of extended Aα-chain as compared to 610/625 Aα-chaincontaining fibrinogen. Protein analysis using SDS-PAGE and Westernblotting analysis indicate that the recombinant fibrinogen is producedin intact format, with the α-chain having the expected size (FIG. 1).

Example 3 Purification of α-Extended Fibrinogen from PER.C6 Cell-CultureMedium

Recombinant human α-extended fibrinogen was purified from cell culturesupernatant according to standard methods. Briefly, (NH₄)₂SO₄ was addedto the culture supernatant to 40% saturation and the precipitate wascollected by centrifugation. Subsequently, the precipitate was dissolvedin TMAE loading buffer (5 mM Tris-HCl pH 8.5, 0.01% Tween-20) followedby dialysis to the same buffer. The protein solution was then loaded ona Fractogel EMD TMAE (m) 40-90 μm (3 ml) (Merck KGaA, Darmstadt,Germany) Ion Exchange Column. Recombinant human α-extended fibrinogenwas subsequently eluted using a continuous salt gradient of 0-1 M NaClin 20 column volumes.

Recombinant human fibrinogen in the peak fractions was precipitatedagain by adding (NH₄)₂SO₄ to 40% saturation and collected bycentrifugation. Finally the material was dissolved in TBS (50 mMTris-HCl, pH7.4, 100 mM NaCl) and dialysed against TBS to remove anyremaining (NH₄)_(SO4) 4.

Example 4 ROTEM Analysis

Clotting time (CT), clot formation time (CFT) and clot firmness of α-extrhFib and plasma derived fibrinogen were measured using ROTEM analysis.ROTEM® (Pentapharm GmbH, Munich, Germany) stands for ROtationThromboElastoMetry. The technique utilizes a rotating axis submerged ina (blood) sample in a disposable cuvette. Changes in elasticity underdifferent clotting conditions result in a change in the rotation of theaxis, which is visualized in a thromboelastogram, reflecting mechanicalclot parameters (see e.g. Luddington R. J. (2005) Clin Lab Haematol.2005 27(2):81).

Pooled normal (citrate) plasma was mixed 1:1 with plasma-derivedfibrinogen (Haemocomplettan, CSL Behring GmbH, Marburg, Germany) orPER.C6 fibrinogen (HMW rhFib or α-ext rhFib) all at 2 mg/ml in TBST(TBS+0.001% Tween-20). Also the fibrinogen preparations (at 2 mg/ml inTBST) were used directly. CaCl₂ was added to a final concentration of 17mM (measurement in plasma) or 1.7 mM (measurement in buffer). To startclotting, α-thrombin was added to a final concentration of 1 IU/ml.ROTEM® analysis graphs for the fibrinogen preparations in buffer ormixed 1:1 with plasma are shown in FIG. 2. A10, A20, CFT and MCF values(mm) are shown in Table 1. A10 and A20 reflect the firmness of the clotat 10 and 20 minutes post α-thrombin addition. CFT reflects the timefrom initiation of clotting until a clot firmness of 20 mm is detected.MCF reflects maximum clot firmness.

The results indicate that clotting of α-ext rhFib in buffer results in astronger clot (A10=12 mm) than what is observed for HMW rhFib (A10=4mm). Plasma derived fibrinogen has an A10 of 15 mm; this stronger clotformation in buffer can be explained by the activity of co-purifiedblood derived FXIII which will (partially) cross-link the fibrinmonomers. In purified recombinant fibrinogen, no FXIII is present andhence no cross-linking can occur. This can be compensated for by runningthe experiment in diluted plasma (which contains FXIII). In this case,stronger clots are formed. Interestingly, in this situation, α-ext rhFibforms a stronger clot and forms it faster than HMW rhFib or plasmaderived Fib, with A20 values of 21, 18 and 17 mm, resp.

TABLE 1 CT A10 A20 MCF CFT Sample (sec) (mm) (mm) (mm) (sec)Plasma-derived 47 10 10 10 — fbg in buffer Plasma-derived 50 15 17 184696 fbg in plasma PER.C6 α-625 70 4 4 4 — fbg in buffer PER.C6 α-625 6117 18 20 3368 fbg in plasma PER.C6 α-847 49 12 12 12 — fbg in bufferPER.C6 α-847 62 18 21 21 950 fbg in plasma

Example 5 Plasmin Digestion of α-ext rhFib and Plasma Derived Fibrinogen

Fibrinogenolysis of purified recombinant human α-ext rhFib was tested byincubation with plasmin. Briefly, fibrinogen was diluted in TBST (50 mMTris-HCl, pH7.4, 100 mM NaCl, 0.01% Tween-20), CaCl2 or EDTA was added(5 mM final concentration) and plasmin was added (10 nM finalconcentration), followed by an incubation at 37° C. At several points intime samples were taken and mixed immediately with SDS-PAGE samplebuffer (NuPAGE LDS sample buffer, Invitrogen, cat# NP0007). Samples werethen subjected to size separation on a non-reduced SDS-PAGE gel (NuPAGE3-8% Tris-Acetate, Invitrogen, cat# WG1602). Protein was visualized byCoomassie staining (SimplyBlue SafeStain, Invitrogen, cat# LC6060).

The results, as shown in FIG. 3, indicate that the plasmin mediateddigestion of α-ext rhFib is significantly slower than for plasma derivedfibrinogen. For example, in the presence of Ca2+, after 60 minutes allof the plasma derived fibrinogen is degraded down to fragment D and Especies. For α-ext rhFib this takes more than 120 minutes and only theovernight incubation shows substantial amounts of fragment E generation,which are already present in the digest of the plasma derived fibrinogenafter 30 minutes.

What is claimed is:
 1. A pharmaceutical human fibrinogen preparation indry form comprising at least 95% (w/w) fibrinogen based on total proteinand wherein at least 10% (w/w) of the fibrinogen is in the form ofFib420, wherein the Fib420 is prepared by a method comprising the stepof: a) providing an expression vector comprising a nucleotide sequenceencoding an alpha extended polypeptide chain of fibrinogen; b)transforming a mammalian cell with the expression vector; and c)maintaining the mammalian cell under such conditions which allow for theexpression of the nucleotide sequence encoding the alpha extendedpolypeptide chain of fibrinogen.
 2. The pharmaceutical human fibrinogenpreparation of claim 1, which is free of Factor XIII.
 3. Apharmaceutical composition comprising the human fibrinogen preparationof claim 1 and a pharmaceutically acceptable carrier, diluents orexcipient.
 4. The pharmaceutical composition of claim 3, wherein thecomposition is suitable for facilitating tissue adherence, improvingwound healing or for intravenous administration.
 5. The pharmaceuticalcomposition of claim 3, wherein the pharmaceutical composition is afibrin sealant or a topical haemostat.
 6. The pharmaceutical compositionof claim 3 which further comprises thrombin.
 7. The pharmaceuticalcomposition of claim 3 in dry form.
 8. The pharmaceutical humanfibrinogen preparation of claim 1 for use as a medicament.
 9. Thepharmaceutical human fibrinogen preparation according to claim 1 for usein a method for treating acute bleeding episodes, hyperfibrinolysis,fibrinogen deficiency or other bleeding disorders.
 10. A method forpreparing a Fib420 fibrinogen preparation, which method comprises: a)providing an expression vector comprising a nucleotide sequence encodingan alpha extended polypeptide chain of fibrinogen; b) transforming amammalian cell with the expression vector; and c) maintaining themammalian cell under such conditions which allow for the expression ofthe nucleotide sequence encoding the alpha extended polypeptide chain offibrinogen.
 11. The method of claim 10, wherein the nucleotide sequenceencoding an alpha extended polypeptide chain of fibrinogen is anoptimized sequence, preferably an optimized sequence according to SEQ IDNo.
 5. 12. The method of claim 10, wherein the mammalian cell is a humancell, preferably a PER.C6 cell.